Along with the progress in diagnostic or medical technology, various methods of detection of particular substances contained in very minute quantities in serum and other biological specimens have been developed and put into use for enabling the early discovery of various diseases and for confirming the effects of therapy. The particular substances to be examined are, for example, various types of proteins, nucleic acids, drugs, and other biological substances. For the purpose of quantitative or qualitative examination of the same, a label as a signal generating source is attached in advance to substances having affinity with such particular substances to be examined, for example, antibodies and antigens where the substance to be examined is a protein; complementary nucleic acids where the substance to be examined is a nucleic acid; and an antibody where the substance to be examined is a drug. The labeled substance having affinity with the substance to be examined and the sample containing the substance to be examined are brought into contact with each other to produce a conjugate of the substance to be examined and the labeled substance having affinity therewith. Then, the conjugate is separated by various methods, the signal generating source of the label is activated, and the signal is detected by various means. As a result, the amount or the existence of the substance to be examined can be determined.
Numerous substances have been developed and supplied for practical use as such a labeling substance, for example, radioactive substances, fluorescent substances, enzymes, or metal colloids. In recent years, however, the chemiluminescence method using an acridinium derivative has drawn attention in view of its high sensitivity. Strong luminescence of the acridinium derivative can be generated by reaction with hydrogen peroxide (H.sub.2 O.sub.2) under strong alkaline conditions (EP-A 830629 etc.)
An attempt has also been made to electrochemically emit light from the acridinium derivative (Anal. Chem. 64, 1140, 1992). According to this report, acridinium derivatives are electrochemically inert, but when a potential of -1.0V (vs. Ag/AgCl) is applied to the electrode under alkaline conditions (pH=12), the dissolved oxygen is reduced to produce hydrogen peroxide. The resulting hydrogen peroxide is reacted with acridinium derivative to generate luminescence. The means for electrochemically emitting light from a chemiluminescent substance is one of the key techniques for realization of immunosensors. More particularly, there is a possibility to be able to simply generate luminescence only by applying a potential to an electrode incorporated in the sensor as a means for generating luminescence of a chemiluminescent substance. The above technique is advantageous to small-sized devices such as sensors. To generate luminescence of an acridinium derivative, however, two steps are required as mentioned above; first rendering to the strongly alkaline conditions, then applying a potential to the electrode. In practice, problems remain to be solved in applying the above technique to an immunosensor.
In the meanwhile, there is a method of semiquantitatively detecting such a substance to be examined, namely so-called blotting, wherein a sample containing the substance to be examined is treated with electrophoresis or the like to separate the substances therein; the separated substances are transferred to and adsorbed on a nitrocellulose film or the like as they are; a labeled substance carrying a substance as a signal generating source and having affinity with the substance to be examined is bonded with the substance to be examined which had been separated and adsorbed on the film, and then the signal generating source is activated to detect the substance to be examined. In this method, it is also possible to detect a substance with an extremely high precision, using chemiluminescence as the detecting means. In the past, however, an enzyme was used as the signal generating source in many cases, and luminol or an adamantane derivative (AMPPD) which can generate luminescence by an enzymatic reaction was used as the luminescent substance. That is, light is emitted from the luminescent substance by carrying out an enzymatic reaction of the signal generating source, and a photosensitive film is exposed with the emitted light to detect the signal.
A luminescent substance was not directly used as a signal generating source in blotting in the past, because the luminescence ends in several seconds when the luminescent substance is used as the signal generating source, and thus an amount of light sufficient to expose a photosensitive film cannot be obtained. Therefore, highly sensitive detection was performed by means of a method wherein an enzyme was used as the signal generating source, and an excess amount of a luminescent substance capable to generate luminescence by the enzymatic reaction therewith was charged so as to emit light for a somewhat long period of time and expose the photosensitive film. Nevertheless, no suitable enzyme capable of generating luminescence by an enzymatic reaction was known for acridinium derivatives in the past. Thus, acridinium derivatives were not used in this field, although having a higher yield of luminescence in comparison with luminol or the like.
The reason why a suitable enzyme was not known is the mechanism of luminescence of the acridinium derivatives. For example, it is known that an acridinium derivative generates luminescence by the reaction with hydrogen peroxide, so it may be considered that luminescence can be generated using an enzymatic reaction, that is, a reaction between H.sub.2 O.sub.2 produced by an enzymatic reaction of oxidase such as glucose oxidase and an acridinium derivative. Unless the acridinium derivative is under strong alkaline conditions, however, the acridinium derivative does not strongly emit light. No enzymes are known which retain their sufficient activity and produce hydrogen peroxide under such strong alkaline conditions.
Further, it is known that an acridinium derivative generates less luminescence under strong alkaline conditions. Accordingly, even if there existed a suitable enzyme which could retain sufficient activity under strong alkaline conditions, there was still the problem that it would not be possible to make sufficient use of the luminescent ability inherently possessed by an acridinium derivative, under luminescent conditions of strongly alkaline conditions for a long term.
As explained above, there was the problem that an acridinium derivative had to be used under strongly alkaline conditions in the conventional light-emitting techniques, so luminescence could not strongly be generated. Therefore, there were considerable limits to its application, despite the high yield of luminescence.
The present inventors engaged in intensive research on improvements of the light emitting method of acridinium derivatives to broaden the application fields thereof, whereupon the inventors surprisingly discovered that strong luminescence is generated even around the neutral condition by reacting an acridinium derivative with the one-electron reductant of dissolved oxygen, a superoxide anion (O.sup.2-). The mechanism of the above luminescence is based on a principle completely different from that of the conventional chemiluminescent method wherein hydrogen peroxide is used under strongly alkaline conditions. Further, because luminescence is generated around neutral condition, the various problems in the prior art can be solved all at once.